A somewhat vexing problem with DNA-directed RNAi (ddRNAi)
has been the heterogeneous mixture of small RNAs generated from a typical expression vector. Not only will
this compromise knockdown efficacy through competition for RISC loading, it also poses a safety risk by increasing
the number of off-target genes. For a
long time, heterogeneous ddRNAi trigger processing had been accepted as a way
of life- as long as the efficacy was right you would not waste too many
thoughts on those multiple bands on your Northern blot.
Choosing the double-strand length of a ddRNAi trigger has
also been more art than science.
Different sizes, usually between 19 and 29bp facilitate potent gene
knockdown, but there have been few studies looking at the consequence of size on the uniformity of hairpin
processing.
Next-generation sequencing technology is changing these
attitudes. In a paper that appeared last
week in CELL, Gu et al. from the Kay lab in Stanford
showed that next-gen sequencing is a powerful tool to detect which small RNA
species are generated from a given ddRNAi template plus their relative quantities. Moreover, through an iterative process of
structural change and sequencing, a key structural feature causing the Dicer enzyme make just one predictable
cut could be identified. Without going
into much detail, a simple 21 base-pair hairpin yields the purest results.
Commercial ddRNAi
trigger landscape getting rusty
While the clinical validation of SNALP delivery technology
has allowed synthetic RNAi trigger-based Tekmira and Alnylam to turn around
their fortunes and should also increase general interest in the technology,
there seem to be no commercial players left in the ddRNAi Therapeutics arena
able to champion and refine the platform.
Benitec is pre-occupied with trying to monetize IP that is
rapidly losing in value due to its age and as a result failing to become a real
biotechnology company with a lab. The two viral delivery companies that
once had some ddRNAi ambitions, AMT (AAV) and Oxford Biomedica (lentivirus),
either went out of business and/or lack the requisite RNAi molecular biology
expertise. And the other groups
developing ddRNAi Therapeutics, including Calimmune and Genables are de facto one-product, disease-focused
companies. This is unfortunate as there
should be room for at least one or two ddRNAi Therapeutics platform companies.
Such a company would have expanded on the basic dsRNA
concept and would thus have kept its IP estate fresh,
eventually forcing others to take a license as simply running out the patent
clock would not have been an option.
Such IP would include discoveries like the one made by Gu and
colleagues. On a more positive note, it is possible that the current revival of gene therapy after a decade of neglect and scorn will eventually carry ddRNAi Therapeutics along with it.
